Effect of Metal Ions on Melanin – Local Anaesthetic Drug Complexes

The affinity of melanin biopolymers for metal ions, drugs and other organic compounds is an important factor in the etiology of toxic retinopathy, hiperpigmentation, otic lesions and irreversible extrapyramidal disorders. The aim of the presented work was to examine the interaction of local anaesthetic drugs used in ophthalmology with model DOPA-melanin in the presence of metal ions. It has been demonstrated that the analyzed drugs form complexes with melanin biopolymer. Based on the .values of association constants,, the following order of drugs affinity to melanin was found: tetracaine > procaine >> bupivacaine > lidocaine. It has also been shown that Cu2+ and Zn2+ ions administered to DOPA-melanin before complexing with drugs decrease the total amount of local anaesthetics bound to melanin. The blocking of some active centers in melanin molecules by metal ions, which potentially exist in living systems, may change the clinical therapeutic efficiency of the analyzed local anaesthetic drugs.


INTRODUCTION
Melanins are biopolymers synthetized in melanosomes of the melanocyte /1,2,3/. The pigmentation system is genetically controlled at multiple levels, including migration of melanocytes during embryogenesis *Corresponding author: Ewa Buszman, Medical University of Silesia, School of Pharmacy, Department of Bioinorganic Chemisoy and Applications Metal ions-melanin complex formation Dry DOPA-melanin samples of 300rag each were mixed with 300ml of bidistilled water containing 1.103M concentration of Cu 2+ (CuCI2 2H_O, PPH POCH, Poland) or Zn z+ (ZnCIz, Merck, Germany). The mixtures were incubated at room temperature for 24h and then filtered. The amounts of copper and zinc bound to melanin were determined by the use of atomic absorption spectrophotometer type AAS 3 (Carl Zeiss, Jena). The final metal ions-DOPA-melanin complexes contained 0.43pmol Cu+/mg melanin and 0.30tmol Zn2+/mg melanin.
Drug-melanin complexes were obtained as follows: 5mg of melanin or metal ion-melanin complexes were placed in plastic test-tubes, where drug solutions in phosphate buffer were added to a final volume of 5ml.
The initial concentration of drugs ranged from o104M to 1.103M. Control samples contained 5rag of melanin and 5ml of buffer without drug. All samples were incubated for 24 h at room temperature, and then filtered.

Analysis of drug binding to melanin
The concentrations of drugs remaining in each filtrate after incubation with melanin were determined spectrophotometrically. All spectrophotometric measurements were made using the UV-VIS spectrophotometer Jasco model V-530. The amounts of drug bound to melanin, calculated as the difference between the initial amount of drug administered to melanin and the amount of unbound drug (in filtrate after incubation) were expressed in lumoles of bound drug per mg melanin. The complex formation efficiency (in %) was also determined as the ratio of drug (lumole) bound to melanin to the total amount of drug (pmole) added to melanin, multiplied by 100. A qualitative analysis of drug-melanin interactions was performed using the Scatchard plot of the experimental data according to Kalbitzer and Stehl ik /3 0/. The number of binding sites (n) and the values of association constant (K) were calculated.

RESULTS
The effect of metal ions on local anaesthetics-melanin complex formation efficiency is presented in Table  1. The affinity of lidocaine and bupivacaine to melanin and metal ion-melanin complexes was analyzed as described in the Experimental section, whereas the results for procaine and tetracaine complexes are known from previous studies/31/. As shown in Table 1, the two-fold increase of initial drug concentration (from 5.10aM to 1-103M) results in the decrease of percentage of drug bound to melanin or metal ion-melanin complex. Simultaneously, the increase of the absolute amount of local anaesthetics bound to melanin (in Effect of.Metal lons on Melanin-Local Anaesthetic Drug Complexes Table   Binding of local anaesthetics to DOPA-melanin as well as to metal ions-melanin complexes". bResults from previous studies in the lab/31/ gmoles per mg melanin) with the increase of initial drug concentration has been demonstrated ( Fig. 1A). Cu z+ and Zn z+ ions fixed by melanin before complexing with drug modify the local anaesthetics binding ability. The decrease of the amount of drug bound to melanin in the presence of metal ions was observed, especially in the presence of Znz, as compared with drug-melanin complexes obtained in the absence of metals.
The experimental data were also analyzed by constructing the Scatchard plots to determine the binding sites and the number of relevant binding classes. For lidocaine-melanin and bupivacaine-melanin complexes the Scatchard plots are linear (Fig. B), indicating that one class of binding sites participates in the formation of these complexes. For procaine-melanin and tetracaine-melanin complexes the Scatchard plots were found to be curvilinear/31/, which indicates that more than one binding class was involved. The calculated binding parameters for the interaction of local anaesthetics with melanin and metal ion-melanin complexes are shown in Table 2. The values of association constant for lidocaine-melanin and bupivacaine-melanin complexes (K103M) demonstrate that mainly weakly reacting sites exist in these complexes. For procaine-melanin and tetracaine-melanin interactions strong binding sites with the association constant KI05M and weak binding sites with KzI03Mwere stated. In the presence of Cu 2+ and Zn 2+ ions an approximately two-fold Coinitial drug concentration, Caconcentration of unbound drug decrease of the number of binding sites (n) was observed for lidocaine and bupivacaine interaction with melanin. For procaine and tetracaine complexes with melanin the decrease of total binding capacity (ntot) in the presence of metal ions was also demonstrated. The presented results indicate that Cu 2+ and Zn 2 ions complexed with melanin modify the binding ability of local anaesthetic drugs by blocking some active centers in the melanin molecules.

DISCUSSION
The eye is located externally i the body and is vulnerable to injury and exposure to toxic environmental insults. Despite its small mass, it carries out biochemical processes which require an avid and diverse blood supply. This results in this organ being second only to the liver in showing manifestations of drug toxicity especially from compounds present in the systemic circulation/18/.
Optical measurements of the pigments of the retinal pigment epithelium (RPE) and choroid have shown that choroidal melanin was always greater than that in the RPE. Simultaneously, it was found that the content of both choroidal and RPE melanin showed a trend of decreasing with aging/32/. Prota et al. /33/ have stated that in human irides both eumelanin and pheomelanin are present, which indicates that differences in stromal pigmentation may be due not only to the quantity, but also to the nature of melanin pigment. It has also been suggested that the differences between human brown and blue eyes in their melanin content may have relevance to the pharmacokinetics of drugs that bind to melanin. This would mean that the larger amounts of melanin would decrease the initial levels of the .drugs and would increase the drug levels after prolonged periods/34/.
Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of metal ions and drugs, and, therefore, serve as a versatile partner of the retinal pigment epithelium (RPE) cell. New findings show that the melanin granules are connected to the iysosomal degradation pathway. Most of these functions are not yet understood, but it was demonstrated that the deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness/35/.
At present, many drugs are known to be markedly accumulated and retained for a considerable time by pigmented tissues and that the retention of these compounds is proportional to the degree of melanin pigmentation/10/. The ability of melanins to bind different drugs and transition metal ions is probably of the greatest biological importance/10,20,25,36/.
Many previous studies have focused on the ability of a compound to bind to melanin in vitro as a measure of its affinity for melanin-containing structures in vivo. The experimental approach often entails the isolation of melanin from biologic sources or the preparation of synthetic melanin from its precursors/37/. Synthetic melanins prepared enzymatically or chemically from L-DOPA contain more carboxyl groups than natural melanins /12/. Furthermore, one can assume that the ion-exchange, redox and fi'ee radical properties established in synthetic melanin are also relevant for natural melanins /38/. Both natural and synthetic melanin polymers have been used in binding studies of ligands and no significant differences in the affinity were observed/12,39/. The earlier experiments with synthetic melanin have shown that metal ions had the same order of affinity for synthetic melanin as for eye-melanin/20/. This suggests that the protein moiety of the melanin plays a minor role in the binding of metal ions. However, a high affinity of metal ions for melanin in vitro does not necessarily mean that a high uptake occurs also in vivo. Melanin is an intracellular constituent, and transport barriers at the cell membranes may prevent ions fi'om reaching the melanin in vivo /20/.
More complete analysis of the metal ions-melanin interaction was performed earlier in our laboratory /36,40,41/. It was shown that two classes of independent binding sites participate in the interaction of Fe , ions with synthetic and natural melanin have been identified and a role of melanin carboxyl, phenolic hydroxyl, amine and imine groups has been discussed. It has been also reported that binding of metal ions to melanin is a time-dependent process/38,41/. The effect of metal ions on drug binding ability to melanin was examined previously 140/. It has been demonstrated that in the interaction of chloroquine with synthetic DOPA-melanin two classes of independent binding sites must be implicated. Metal ions (Cu2+, Zn2+, Mn2+, Co2+, Ni2+) fixed by melanin polymer before complexing with drug are the blocking agents, because only one class of binding sites has been found for chloroquine-(metal ion-melanin) complexes 1431. It has also been shown that metal ions incorporated into melanin during its synthesis modify the drug binding ability/44,451.
The results presented in this study indicate that local anaesthetics used in ophthalmology form complexes with melanin biopolymer. Based on the values of association constants, the following order of drugs affinity to synthetic DOPA-melanin was found: tetracaine > procaine >> bupivacaine >lidocaine. It has also been demonstrated that Cu 2+ and Zn 2+ ions administered to DOPA-melanin before complexing with drugs decrease the total amount of local anaesthetics bound to melanin. The nature of the melanin-ligand interactions is still not well established but the existence of ionic bonds, Van der Waals forces, hydrophobic interactions and change-transfer reactions in drug-melanin complex formation has been suggested/10/. Taking into account that metal ions as well as the analyzed drugs exist in the form of cations at physiological pH, probably the same active centers in melanin polyanion are responsible for these ligands binding. The blocking of some active centers in melanin molecules by metal ions which potentially exist in living systems may change the clinical therapeutic efficiency of the analyzed local anaesthetic drugs.